Accumulation Dynamics of Transcripts and Proteins of Cold-Responsive Genes in Fragaria Vesca Genotypes of Differing Cold Tolerance

International Journal of Molecular Sciences

Article Accumulation Dynamics of Transcripts and Proteins of Cold-Responsive Genes in Fragaria vesca Genotypes of Differing Cold Tolerance

Isam Fattash 1,*, Zachary Deitch 2, Relindis Njah 3, Nelson Osuagwu 4 , Vera Mageney 5, Robert C. Wilson 3, Jahn Davik 6, Muath Alsheikh 7,8 and Stephen Randall 2

1 Department of Biology and Biotechnology, American University of Madaba, Madaba 11821, Jordan 2 Biology Department 723 W Michigan Street IUPUI, Indianapolis, IN 46202, USA; [email protected] (Z.D.); [email protected] (S.R.) 3 Department of Biotechnology, INN University, 2318 Hamar, Norway; [email protected] (R.N.); [email protected] (R.C.W.) 4 Department of Clinical Medicine, University of Bergen, Postboks 7804, N-5020 Bergen, Norway; [email protected] 5 Institute for Biology and Environmental Sciences (IBU), Carl von Ossietzky Universität Oldenburg, Carl von Ossietzky-Str. 9-11, 26111 Oldenburg, Germany; [email protected] 6 Department of Molecular Plant Biology, Norwegian Institute of Bioeconomy Research, Høgskoleveien 8, N-1433 Ås, Norway; [email protected] 7 Graminor Breeding Ltd., Hommelstadveien 60, N-2322 Ridabu, Norway; [email protected] 8 Department of Plant Sciences, Norwegian University of Life Sciences, P.O. Box 5003, 1432 Ås, Norway * Correspondence: [email protected]

Citation: Fattash, I.; Deitch, Z.; Njah, R.; Osuagwu, N.; Mageney, V.; Abstract: Identifying and characterizing cold responsive genes in Fragaria vesca associated with or Wilson, R.C.; Davik, J.; Alsheikh, M.; responsible for low temperature tolerance is a vital part of strawberry cultivar development. In this Randall, S. Accumulation Dynamics study we have investigated the transcript levels of eight genes, two dehydrin genes, three putative of Transcripts and Proteins of ABA-regulated genes, two cold–inducible CBF genes and the alcohol dehydrogenase gene, extracted Cold-Responsive Genes in from leaf and crown tissues of three F. vesca genotypes that vary in cold tolerance. Transcript levels of Fragaria vesca Genotypes of Differing the CBF/DREB1 transcription factor FvCBF1E exhibited stronger cold up-regulation in comparison Cold Tolerance. Int. J. Mol. Sci. 2021, to FvCBF1B.1 in all genotypes. Transcripts of FvADH were highly up-regulated in both crown and 22, 6124. https://doi.org/10.3390/ leaf tissues from all three genotypes. In the ‘ALTA’ genotype, FvADH transcripts were signiﬁcantly ijms22116124 higher in leaf than crown tissues and more than 10 to 20-fold greater than in the less cold-tolerant ‘NCGR1363’ and ‘FDP817’ genotypes. FvGEM, containing the conserved ABRE promoter element, Academic Editor: Achim Aigner transcript was found to be cold-regulated in crowns. Direct comparison of the kinetics of transcript and protein accumulation of dehydrins was scrutinized. In all genotypes and organs, the changes Received: 9 April 2021 Accepted: 27 May 2021 of XERO2 transcript levels generally preceded protein changes, while levels of COR47 protein Published: 7 June 2021 accumulation preceded the increases in COR47 RNA in ‘ALTA’ crowns.

Publisher’s Note: MDPI stays neutral Keywords: dehydrins; ABA-regulated genes; CBF genes; alcohol dehydrogenase with regard to jurisdictional claims in published maps and institutional afﬁl- iations. 1. Introduction Abiotic stress such as cold is a serious threat to the sustainability of agricultural productivity, causing crop loss worldwide, signiﬁcantly reducing average yields for most major Copyright: © 2021 by the authors. crops [1]. The diploid woodland strawberry (Fragaria vesca) is an herbaceous perennial plant Licensee MDPI, Basel, Switzerland. that grows naturally in most regions of the northern hemisphere. F. vesca (2n = 2x = 14), This article is an open access article is a versatile experimental plant with a small genome (240 Mb). It has a robust genetic distributed under the terms and transformation system [2] and shares high sequence similarity with the cultivated straw- conditions of the Creative Commons berry (Fragaria × ananassa) and other members of the Rosaceae family. F. vesca was selected Attribution (CC BY) license (https:// for sequencing as a reference genome for the Rosaceae because of its advantages over creativecommons.org/licenses/by/ other family members, including a short generation time for a perennial, ease of vegetative 4.0/).

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propagation and small herbaceous stature compared with tree species such as peach or apple [3]. The strawberry plant is highly variable in susceptibility to freezing injury, one of the largest factors affecting crop yield and quality in temperate regions. Winter damage to strawberry plants can result in losses of up to 50% annual yield in production in Scandi- navia [4]. Overwintering success relies on the crown to remain unaffected not only by the physical damage induced by freezing but also to survive for extensive periods of time at low sub-zero temperatures, and to be optimally resistant to fungal and bacterial invasion favored by freezing induced damage [5]. Thus, one of the major objectives of a strawberry breeding program is to develop cultivars that are winter hardened and highly cold and freezing tolerant. Identification of molecular markers associated with, or responsible for, low temperature tolerance could be employed in strawberry breeding programs [6]. Low temperature (LT) causes oxidative stress, which affects both photosynthetic and respiratory mechanisms in plants. It slows down enzymatic reactions which affect the demand for ATP, leading to an overflow of electrons and an increase in the level of reactive oxygen species (ROS) including superoxide anions, hydroxyl radicals and hydrogen per- oxide [7]. Oxidative stress that results from extreme low temperature (freezing) inhibits the activities of the enzymes glutathione reductase, dehydroascorbate reductase and mon- odehydroascorbate reductase that protect the plants against ROS [8]. Cold stress in plants triggers the activity of a number of genes, which alter the level of metabolites and proteins that are responsible for providing protection against the stress. Cold stress signals are transduced through ABA-dependent and ABA-independent pathways [9–11]. In addition to the role of the phytohormone abscisic acid (ABA) in various plant developmental processes, such as leaf abscission and initiation of fruit ripening; it is also an important contributor to responses to abiotic stresses. ABA-induced gene expression is mediated by the presence of cis-acting elements known as ABA responsive elements (ABREs) [12,13]. The ABA-dependent cold pathway activates genes with ABREs in their promoters, while an ABA-independent (CBF/DREB1) cold pathway activates genes with CRE/DREs in their promoters [14,15]. Transcription factors called the C-repeat binding factors (CBFs) or dehydration-responsive element-binding factors (DREBs) have been identified as major contributors to responses leading to cold/freezing tolerance in Arabidop- sis thaliana [16] and are indeed essential to develop optimal cold/freezing tolerance [17]. The signaling pathway by which CBFs become activated has been extensively characterized [9,18–20]. It is clear that other transcription factors have important contributory roles in the cold response [11,21]. The downstream targets that are coordinately regulated by CBFs include hundreds of genes [22]. Among those are alcohol dehydrogenases (ADH) and dehydrins. Interestingly, these genes can be regulated by both ABREs and CRE/DREs. Alcohol dehydrogenase (ADH) is a Zn-binding oxidoreductase that depends on NAD(P)H for interconversion of ethanol and acetaldehyde. ADH is thus associated with alcoholic fermentation where it produces a relatively nontoxic end product of anaerobiotic glycolysis contributing ATP and a small amount of NAD+ that can support glycolysis. Chilling tolerant species are induced towards anaerobic metabolism during LT, which results in increased ADH expression [23]. Increases in ADH protein levels also occur in response to cold in both diploid [24] and octoploid species of strawberry [5]. In other species, expression of alcohol dehydrogenase (ADH) is known to increase under various stresses, including low temperature, drought, abscisic acid (ABA) and salinity [25–31]. In particular, ADH genes are among the most commonly found cold-induced genes in cereal crops and Arabidopsis [30]. However, knockout experiments suggest that the single ADH gene in Arabidopsis is not required for freezing tolerance [26]. Dehydrins (DHNs) belong to a large group of highly hydrophilic proteins known as late embryogenesis abundant (LEA) proteins. A unique feature of all DHNs is a conserved, lysine-rich 15-amino acid domain, EKKGIMDKIKEKLPG [32], the K-segment, which is usually located at the C-terminus with a putative ability to form an amphipathic helix structure that can interact with membrane proteins [33]. Other common features of DHNs are a tract of serine residues (the S-segment), which is part of a conserved motif Int. J. Mol. Sci. 2021, 22, 6124 3 of 21

(LHRSGS4-10(E/D)3) that undergoes phosphorylation and may regulate ion-binding and protein conformation [34,35]. The consensus motif DEYGNP (the Y-segment) found in some dehydrins is located near the N-terminus [36]. DHNs are often classified according to the number and arrangements of these conserved K-, Y- and S- segments [36–38]. Evidence supports several proposed physiological functions of dehydrins, including responding to low temperature, drought, salt and heavy metal binding [24,39–43]. In vitro and in vivo studies have shown that some DHNs bind to ions and metals thereby potentially reducing toxicity [44] or the associated production of oxygen free radicals [2,35,45,46]. They may also function as a cryoprotective substance towards freezing sensitive enzymes [47]. While some dehydrin proteins accumulate in seeds late in embryogenesis, distinct dehydrins are present in nearly all the vegetative tissues during normal growth conditions and are highly accumulated in response to abiotic stress [37]. The vegetatively expressed dehydrins in A. thaliana are the glutamic acid-rich, acidic DHNs which include COR47, ERD10 and ERD14 and the glycine-rich, neutral/basic DHNs: RAB18, XERO1, and XERO2 [35,37]. Overex- pression studies of DHNs have shown the positive effects of dehydrin gene expression in enhancing cold tolerance in plants [48–50] and particularly in octoploid strawberry [51]. Octoploid strawberry responses to LT include alterations in protein expression and metabolites [5,43,52] which provides enhanced levels of antioxidants, disease resistance proteins and molecular chaperones, as well as protective metabolites such as amino acids, sugars, galactinol and raffinose [43,52]. In F. vesca diploids, the protein levels of alcohol dehydrogenase and total dehydrins correlate highly with the degree of tolerance to extensive freezing over a number of distinct genotypes [24]. To examine responses in differentially cold-tolerant plants, diploid strawberry genotypes of F. vesca previously characterized as high (‘ALTA’, LT50 = −11.6 ◦C), moderate/low (‘NCGR1363’, LT50 = −8.2 ◦C), or low (‘FDP817’, LT50 = −7.7 ◦C) freezing tolerance [24] were selected for protein and transcript profile analysis. We aimed to identify genes potentially contributory to cold tolerance in F. vesca, by studying the association of transcript level of homologues of genes known to be involved in freezing tolerance, specifically targeting dehydrins, ABA-responsive genes, and CBFs, and further correlating their expression levels (in the case of dehydrins) with protein abundance. The work described here identifies several of the LT-induced dehydrin proteins in F. vesca and compares their expression changes to their encoding transcripts and further extends the evaluation of cold responses to other potential cold responsive genes, characterizing their time-dependent expression, and explores possible mechanisms contributing to cold tolerance.

2. Results 2.1. Identification of Putative F. vesca CBFs, ABA-Responsive Genes and Dehydrins To identify potential C-repeat binding factors (CBF/DREBs), the NCBI database was searched using BLASTp with the consensus sequence for CBFs (in particular the AP2 domain) as query. Twelve potential Fragaria CBFs (AP2 domain-containing proteins) were identified (Table S1) and aligned with Arabidopsis, tomato and soybean CBF/DREB1s (Figure S1). Two Fragaria CBF candidates (FvCBF1B.1, FvCBF1E) grouped in the same clade as the Arabidopsis, tomato and soybean CBF/DREB1s. CBF1B was the most similar of the remaining candidates and was more distantly related to CBF1B.1. The remaining AP2-containing proteins grouped as DREB2s (drought response element binding proteins) or ERFs (ethylene response factors). A comparison of the diagnostic sequences immediately flanking the AP2 DNA-binding domain [53] showed that the FvCBF1B.1 and FvCBF1E were most similar to the functionally characterized CBFs, while CBF1B.2 lacked similarity immediately downstream of the AP2 domain. Thus only FvCBF1B.1 and FvCBF1E possessed the conserved C-terminal DSAWR consensus sequence. Three putative ABA-responsive genes in F. vesca (Table S2) were identified as containing ABREs within 1100 b.p. of their ATGs, (1) GEM-like protein 5-like (XP_004304553.1), (2) BTB/POZ domain-containing protein At5g66560-like (XP_004301873.1) and 3) RING-H2 finger protein Int. J. Mol. Sci. 2021, 22, 6124 4 of 21

ATL11-like (XP_004301345.1). Seven putative F. vesca dehydrins were identiﬁed (Table S2). Arrangements of the K-, S- and Y-segments in these protein sequences are shown in Figure S2 and sequential relationships and a comparison to Arabidopsis dehydrins is shown in Figure S3. The dehydrins of A. thaliana and F. vesca cluster in two major phylogenetic groups. Four of the putative F. vesca dehydrins (protein FvDehydrin, FvXERO1-like, FvDHN2-like and FvXERO2-like) clustered together in one major group and were closely related to the A. thaliana XERO1 and 2, and Rab18 dehydrins. The second major phylogenetic group comprises the COR47 and ERD dehydrins, which includes the two COR47-like (A and B) F. vesca proteins. COR47-like A and COR47-like B dehydrins were most clearly distinguished from each other in predicted protein size (179 aa and 246 aa, respectively). The two identiﬁed Fragaria dehydrins can be categorized [36–38] as SKn (e.g., COR47 A, B) and YnSKn (e.g., XERO2 and CS66-like).

2.2. Expression Analysis Transcript levels of eight genes (two dehydrin genes, FvCOR47B and FvXERO2 belong- ing to the LEA protein family group; three putative ABA-regulated genes, FvGEM, FvBTB and FvRHA11; two cold-inducible CBF genes FvCBF1B.1 and FvCBF1E; and the alcohol dehydrogenase gene FvADH) were analyzed in leaf and crown tissues extracted from the three F. vesca genotypes (‘ALTA’, ‘NCGR1363’ and ‘FDP817’). Changes in transcript levels were compared across the genotypes to estimate relative responses to cold. When interpreting the impact of fold changes, the absolute starting amount of a transcript present across the genotypes must be considered as that, together with cold-induced changes, contributes to the total amount of transcript abundance. There was little variation in basal levels of most transcripts based upon the Cts at time 0 (Table S3). However, the baseline levels of GEM, CBFs, XERO2 and COR47 transcripts were all consistently higher (lower Ct) in the crowns of the ‘ALTA’ genotype, suggesting elevated levels of transcript in the ‘ALTA’ genotype for these genes under nonacclimating conditions.

2.3. Transcript Analysis of FvCBF1B.1 and FvCBF1E The CBF/DREB1 transcription factors are key to cold responses in most cold-tolerant plants, controlling expression of a regulon that consists of more than 100 genes in Ara- bidopsis [54]. Two encoded CBFs, which showed the greatest similarity and clustered in phylogenetic analyses with the Arabidopsis, tomato and soybean cold-responsive CBFs, were examined (Figure S1). FvCBF1B.1 was most similar to these orthologs, occupying the same branch as the Arabidopsis, tomato and soybean CBFs. While FvCBF1E had its own branch, it still clustered with the CBF/DREB1s but not with the DREB2s, which are more involved with drought stress responses. Expression analyses showed a modest but rapid up-regulation of FvCBF1B.1 transcripts in response to cold acclimation (Figure1). Interestingly, the transcript levels for FvCBF1B.1 in ‘ALTA’ crowns were strongly decreased after 1 h, persisting throughout the cold exposure. Across the genotypes, the changes in FvCBF1B.1 transcript levels in crowns were considered quite modest in comparison to other cold responsive plants that showed increases of hundreds- or thousands-fold within 3–6 h [55–58]. Interestingly, changes in FvCBF1B.1 transcript abundance tended to be greater in leaf tissues than in the crowns. Alternatively, FvCBF1E exhibited strong cold up-regulation, peaking in leaves at 3–8 h, typical of a CBF response pattern (Figure1 ). In the crown, a similar pattern was observed but with several hours delay. FvCBF1E transcripts exhibited 100-1000-fold increases in leaf tissue in the two more cold-tolerant genotypes (‘ALTA’ and ‘NCGR1363’), which is usual for cold responsive CBFs in both the leaf and crown tissues. Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 5 of 22 Int. J. Mol. Sci. 2021, 22, 6124 5 of 21

Figure 1. Cold responses of F. vesca CBF transcripts. All transcript levels were normalized to FvPP2A and expressed relative Figureto 1. untreated Cold responses control plants of F. vesca (0 h) inCBF response transcripts. to cold All treatment. transcript The levels crown were and leafnormalized tissues from to FvPP2A three genotypes and expressed ‘ALTA’, relative to‘NCGR1363’ untreated andcontrol ‘FDP817’ plants were (0 coldh) in treated response for 1 to h, cold 3 h, 8treatment. h, 24 h, 48 The h, 7 d,crown 14 d andand 42leaf d, htissues = hours, from d = three days. genotypes PCR ‘ALTA’,performed ‘NCGR1363’ as described, and ‘FDP817’ with respective were cold primer treated pairs (Tablefor 1 h, S4). 3 S.D.h, 8 areh, 24 indicated h, 48 h, as 7 errord, 14 bars. d and 42 d, h = hours, d = days. PCR performed as described, with respective primer pairs (Table S4). S.D. are indicated as error bars. 2.4. Transcript Analysis of FvADH 2.4. TranscriptTranscript Analysis analysis of ofFvADHFvADH showed that this gene was up-regulated at 24 h of LT, generallyTranscript peaking analysis at 48 hof after FvADH the onset showed of cold that (Figure this gene2). In was crowns up of-regulated all genotypes, at 24 the h of LT, generallytranscript peaking was most at 48 highly h after expressed the onset at 48 of h, cold and a(Figure strong decline2). In crowns in transcript of all abundance genotypes, the was observed at 7 d and 14 d followed by a slight increase at 42 d of LT. In the ‘ALTA’ transcript was most highly expressed at 48 h, and a strong decline in transcript abundance genotype, the fold changes were signiﬁcantly higher in leaf than in crown tissues. It was wasinteresting observed to at note 7 d thatand the 14 changesd followed of FvADH by a slighttranscripts increase in both at 42 crown d of andLT. leafIn the in the‘ALTA’ genotype,highly cold-tolerant the fold changes ‘ALTA’ were were significantly much greater higher than in in the leaf other than two in lesscrown cold-tolerant tissues. It was interestinggenotypes, to ‘NCGR1363’ note that the and changes ‘FDP817.’ of FvADH transcripts in both crown and leaf in the highly cold-tolerant ‘ALTA’ were much greater than in the other two less cold-tolerant genotypes, ‘NCGR1363’ and ‘FDP817.’

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Figure 2. Cold responses of F. vesca ADH transcripts. All samples treated as in Figure1. Figure 2. Cold responses of F. vesca ADH transcripts. All samples treated as in Figure 1. 2.5. Transcript Analysis of the Dehydrins, FvXERO2 and FvCOR47 2.5. Transcript Analysis of the Dehydrins, FvXERO2 and FvCOR47 Dehydrin genes are well documented downstream targets of CBFs. We chose to Dehydrin genes are well documented downstream targets of CBFs. We chose to ana- analyze two homologs of dehydrins shown to be cold responsive in other plants (XERO2 lyze two homologs of dehydrins shown to be cold responsive in other plants (XERO2 and and COR47). The FvXERO2 mRNA levels were found to increase with similar kinetics COR47).in ‘ALTA’ The crown FvXERO2 and leaf mRNA (Figure levels3), although were found transcript to increase increases with were similar initiated kinetics slightly in ‘ALTA’earlier in crown the leaves and afterleaf (Figure the starting 3), although of cold treatment. transcript No increases signiﬁcant were accumulation initiated sli ofghtly the

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earlier in the leaves after the starting of cold treatment. No significant accumulation of the Int. J. Mol. Sci. 2021, 22, 6124 XERO2 transcripts were observed until 24 h of LT and the highest accumulation7 of 21 levels were generally found in both leaves and crowns at 42 d of LT. Generally, there was a steady and significant increase in transcript levels from 24 h to 42 d of LT, in crown and leafXERO2 tissuetranscripts of all genotypes were observed at 7 and until 14 24 d h of LTLT. and A thenotable highest exception accumulation was levelsa transient were de- creasegenerally observed found in in both both leavesthe lesser and crownscold-tolerant at 42 d ofgenotypes LT. Generally, (‘NCGR1363’, there was a steady‘FDP817’). and The FvCOR47signiﬁcant transcript increase was in transcript also up levels-regulated from 24in hall to cold 42 d ofacclimated LT, in crown plants and leaf(Figure tissue 3). of In all genotypes,all genotypes both at crown 7 and 14and d ofleaves LT. A showed notable exception a peak accumulation was a transient of decrease FvCOR47 observed at 48 h in of LT. Thisboth transcript the lesser showed cold-tolerant a lower genotypes fold increase (‘NCGR1363’, across ‘FDP817’). all genotype/tissue The FvCOR47-type/timetranscript-point- was also up-regulated in all cold acclimated plants (Figure3). In all genotypes, both crown combinations compared to the FvXERO2 transcripts (approximately 10-fold lower). With and leaves showed a peak accumulation of FvCOR47 at 48 h of LT. This transcript showed a thelower exception fold increase of NCGR1363 across all genotype/tissue-type/time-point-combinations the changes in FvCOR47 transcript levels comparedwere greater to in leavesthe FvXERO2 than in thetranscripts crown tissues. (approximately All genotypes 10-fold lower). showed With similar the exception temporal of NCGR1363expression pat- ternsthe in changes leaves: in aFvCOR47 gradualtranscript increase levelsin transcript were greater accumulation in leaves than from in the1 h crownto 48 tissues.h, followed by Alla decrease genotypes extending showed similar to 42 d temporal of LT. In expression crowns, patternsthough inall leaves: showed a gradual increases increase in FvCOR47 in transcripttranscript levels, accumulation no consistent from 1 hpattern to 48 h, in followed the transcript by a decrease levels extending across genotypes to 42 d of LT.was In obvious.crowns, though all showed increases in FvCOR47 transcript levels, no consistent pattern in the transcript levels across genotypes was obvious.

Figure 3. Cold responses of F. vesca Dehydrin transcripts. All samples treated as in Figure1. Figure 3. Cold responses of F. vesca Dehydrin transcripts. All samples treated as in Figure 1. 2.6. Transcript Analysis of Putative ABRE-Containing FvGEM, FvBTB and FvRHA11 2.6. TranscriptThe expression Analysis levels of Putative of three ABRE genes- containingContaining putative FvGEM, ABA-responsive FvBTB and FvRHA11 elements in theirThe promoters expression were levels examined of three (Figure genes4). containingAtGEM-related putative genes ABA have- ABREs,responsive are ABA elements regulated and are likely functioning as part of the ABA signaling pathway [59]. FvGEM in their promoters were examined (Figure 4). AtGEM-related genes have ABREs, are ABA transcripts were slightly up-regulated in crowns in response to cold treatment in the two regulatedlesser cold-tolerant and are likely genotypes functioning with a gradual as part accumulation of the ABA oversignaling the entire pathway time in [59]. the cold FvGEM tran(upscripts to ﬁve-fold) were slightly (Figure4 up). The-regulated levels of inFvGEM crownstranscript in response were decreasedto cold treatment in cold-treated in the two lesser‘ALTA’ cold genotype-tolerant crown genotypes tissues with and increaseda gradual slightly accumulation in the crowns over ofthe the entire other time genotypes in the cold (upwhile to five no-fold) signiﬁcant (Figure differences 4). The levels were of observed FvGEM in transcript the expression were indecreased leaves between in cold the-treated ‘ALTA’three genotypes.genotype BTB/POZcrown tissues domain and containing increased proteins slightly have in been the showncrowns to of be the responsive other genotypes while no significant differences were observed in the expression in leaves between

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Int. J. Mol. Sci. 2021, 22, 6124 8 of 21 the three genotypes. BTB/POZ domain containing proteins have been shown to be responsive to ABA, modulating seed germination or ethylene responsive pathways [60]. FvRHA11to ABA, is modulating a member seed of the germination ring finger or-containing ethylene responsive ATL (RHA pathways) gene [family60]. FvRHA11 of ubiquitin ligases,is a member some members of the ring of ﬁnger-containing which are ABA-ATLresponsive(RHA) gene [61]. family The ATL of ubiquitin gene family ligases, from A. thalianasome membersand Oryza of whichsativa arecomprises ABA-responsive a large number [61]. The ofATL putativegene family ubiquitin from A. ligases thaliana of the and Oryza sativa comprises a large number of putative ubiquitin ligases of the RING-H2 RING-H2 type [61]. LT treatment generally led to strong decreases in FvBTB and FvRHA11 type [61]. LT treatment generally led to strong decreases in FvBTB and FvRHA11 levels in levelsthe leaves,in the leaves, maximal maximal at 24 and at 48 24 h. Inand crowns 48 h. theIn crowns expression the levels expression of FvBTB levelsand FvRHA11 of FvBTB and FvRHA11were minimal were minimal in most genotype/tissue-type in most genotype/tissue combinations-type combinations (Figure4). (Figure 4).

FigureFigure 4. Cold 4. Cold responses responses of F. of F.vesca vesca potentialpotential ABA ABA responder transcripts. transcripts. All All samples samples treated treated as in as Figure in Figure1. 1. 2.7. Analysis of Total Dehydrin Protein Accumulation 2.7. Analysis of Total Dehydrin Protein Accumulation Having examined the transcriptional response of two dehydrin genes (Figure3) we furtherHaving examined examined the general the transcriptional expression pattern response of the dehydrinof two dehydrin proteins (Figuregenes 5(Figure). Dehy- 3) we furtherdrins examined previously describedthe general as 25 expression kDa, 35–37 pattern kDa and of 60–65 the kDadehydrin (see Supplemental proteins (Figure Material 5). De- hydrinsin [24]) previously were detected described in F. vesca as by25 thekDa, antibodies. 35–37 kDa In and general, 60–65 two kDa patterns (see Su ofpplemental changes in Ma- terialdehydrin in [24]) protein were levels detected in response in F. to vesca LT were by theobserved. antibodies. The first In pattern general, observed two patterns was a of changeslong delay in dehydrin in changes protein of expression, levels in often response no significant to LT were increases observed. seen until The ca. first 7 d (168pattern h) ob- servedof LT, was after a whichlong delay an increase in changes in dehydrin of expression, levels was often detected. no signifi This wascant exhibitedincreases in seen the until anti-AtXERO2-reactive bands in all genotypes in both crowns and leaves (Figure5) and for ca. 7 d (168 h) of LT, after which an increase in dehydrin levels was detected. This was the anti-AtCOR47-reactive bands from FDP817 crowns. The second pattern was an initial exhibitedrapid response in the anti (seen-AtXERO2 within the- firstreactive few hours bands of in LT) all followed genotypes by a in decrease both crowns and then and after leaves (Figure7 d, a 5) strong and increase.for the anti This-AtCOR47 was exhibited-reactive best bybands COR47 from reactive FDP817 bands crowns. in ‘ALTA’ The crownssecond pat- ternand was less an by initial ‘ALTA’ rapid leaves response and ‘NCGR1363’ (seen within leaves the (Figure first 5few). ‘NCGR1363’ hours of LT) crowns followed and by a decrease‘FDP817’ and leaves then showed after 7 nod, significanta strong increase. responses This of COR47 was exhibited reactive proteins best by to COR47 LT. reactive bands in ‘ALTA’ crowns and less by ‘ALTA’ leaves and ‘NCGR1363’ leaves (Figure 5). ‘NCGR1363’ crowns and ‘FDP817’ leaves showed no significant responses of COR47 reactive proteins to LT.

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Figure 5. Cold responses of F. vesca dehydrin protein. (A,B) corresponding fold changes of FvCOR47 protein levels in Figure 5. Cold responses of F. vesca dehydrin protein. (A,B) corresponding◦ fold changes of FvCOR47 protein levels in crownscrowns and and leaves leaves of of three threeF. F. vesca vescagenotypes genotypes in in response response toto coldcold (2(2 C) over time, time, respectively. respectively. (C (C,D,D) are) are graphs graphs of offold fold ◦ changingchanging of FvXERO2of FvXERO2 protein protein levels levels in in crowns crowns and and leavesleaves ofof three F. vesca genotypes in in response response to to cold cold (2 (2C)C) over over time, time, respectively.respectively. All All samples samples were were obtained obtained as as in in Figure Figure1 .1. Note Note that that althoughalthough the linear scale scale on on the the x x-axis-axis is is shown shown in inhours, hours, thesethese are are the the same same samples samples and and timepoints timepoints as as shown shown inin FiguresFigures1 1––44 (i.e., (i.e., 10081008 h time point is is 42 42 d). d). Genotypes Genotypes used used are are designateddesignated as FDPas FDP indicates indicates the the FDP817; FDP817; NCGR NCGR indicates indicates NCGR1363, NCGR1363, and and ALTA. ALTA. Crown Crown tissues tissues are are indicated indicated as as “crn”, “crn”, leaf leaf tissues as “lf”. Each lane in the gels were loaded with the same total protein (10 μg) and dehydrin levels were deter- tissues as “lf”. Each lane in the gels were loaded with the same total protein (10 µg) and dehydrin levels were determined mined following Western blotting and were quantitated as described in Methods. Westerns were probed with either anti- followingCOR47 Westernor anti-XERO2 blotting antibodies and were as quantitated indicated. as described in Methods. Westerns were probed with either anti-COR47 or anti-XERO2 antibodies as indicated. 2.8. Identification of COR47-like and XERO2-like proteins and correlation with their respective 2.8.transcripts Identification of COR47-Like and XERO2-Like Proteins and Correlation with Their Respective Transcripts The accumulation of dehydrin proteins as described above in diploid woodland strawberryThe accumulation is slow relative ofdehydrin to the rapid proteins accumulation as described observed above in other in diploid cold responsive woodland strawberryspecies [24,43]. is slow The relative differential to the expression rapid accumulation in the different observed genotypes in (Figure other cold 5) suggested responsive speciesthat a closer [24,43 ].examination The differential of the expression kinetics of inboth the transcript different and genotypes protein (Figureaccumulation5) suggested was thatnecessary a closer to examination understand their of the possible kinetics contribution of both transcript to the acquisition and protein of cold accumulation tolerance in was necessarystrawberry. to To understand determine their the correspondence possible contribution of specific to dehydrin the acquisition transcripts of cold (qRT tolerance-PCR) inwi strawberry.th their respective To determine dehydrin the proteins correspondence (Western blots), of specific the identity dehydrin of the transcripts dehydrin pro- (qRT- PCR)teins with was theirrevealed respective by mass dehydrin spectroscopy proteins (Figure (Western 6). The blots), dehydrins the identity were purified of the dehydrin from proteins‘ALTA’ was crowns revealed (Figure by 6A). mass Two spectroscopy major bands (Figure and 6one). The minor dehydrins band corresponded were purified with from ‘ALTA’anti-XERO2 crowns and (Figure anti-COR476A). Two activity major were bands eluted and in one a high minor salt band fraction corresponded from an anion with anti-XERO2exchange column and anti-COR47 (Q1000), one activity at 60–65 were kDa, eluted a doublet in a at high approximately salt fraction 37 from kDa anand anion a exchangeminor band column at 25 (Q1000),kDa. The onethree at regions 60–65 kDa,(60–65, a doublet37 and 25 at kDa) approximately were excised 37 from kDa andthe a minorSDS-PAG bandE atgel 25 (the kDa. doublet The threeat 37 kDa regions excised (60–65, as one) 37 and prepared 25 kDa) werefor analysis excised by from mass the SDS-PAGEspectroscopy. gel (the Masses doublet of unique at 37 tryptickDa excised fragments as one) (underlines and prepared in Figure for analysis 6C) consistent by mass spectroscopy.with distinctive Masses single ofdehydrin unique sequences tryptic fragments were obtained (underlines from the in 60 Figure–65 (Figure6C) consistent 6A,B,) with distinctive single dehydrin sequences were obtained from the 60–65 (Figure6A,B) and the 37 kDa (Figure6B) regions of the gel (Figure6C). Band 60–65 kDa was thus identified as COR47-like and the 37 kDa band was identified as XERO2-like. Identification

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of the 25 kDa band was unsuccessful, likely due to the low abundance as indicated by the barely detectable Coomassie-stained band (Figure6B). The changes in protein bands corresponding to XERO2 and COR47 (Figure7A–D ) were then compared to their respective transcripts (Figure8). In all genotypes and tissues, the changes in XERO2 transcript levels generally preceded protein changes (Figure8 and Figure S5). Similarly, COR47, in the leaves of ‘ALTA’, ‘NCGR1363’ and ‘FDP817’ and in the crowns of ‘NCGR1363’ and ‘FDP817’ the changes in COR47 transcripts preceded the changes in COR47 proteins (Figure S4). However, in ‘ALTA’ crowns, the most cold-tolerant of all three genotypes, and the genotype showing the greatest increase and greatest absolute levels of COR47 proteins at 42 d, changes in protein were concurrent or even preceded the increases in transcript levels (Figure8). It is important to mention that the cold-associated changes in COR47 transcript levels in ‘ALTA’ are lowest of all genotypes, yet the protein levels change the most. We found this quite intriguing that the most cold-tolerant genotype had the poorest correlation between LT-induced abundance changes in COR47 protein and transcripts. To further understand the increase of COR47 protein that anticipates the increase in COR47 transcript in ‘ALTA,’ we first considered the unlikely possibility that one of the two isogenes of COR47A and B might be contributing to this interpretation. We considered this unlikely because we only observed a single major antiCOR47 reactive protein band in Western blots (Figure7B); whereas, if both genes were being expressed, one of them (COR47A) would be predicted to be approximately 50% smaller in mass (see Figure S2). Transcripts derived from COR47A and COR47B were not distinguished in earlier qRT- PCR experiments (Figure3), so we developed COR47A and B transcript specific primers (Table S4) and examined the levels of transcripts in ‘ALTA’ crowns. The COR47A gene was expressed at significantly lower levels (>1000 fold lower) than COR47B. Thus, COR47B seemed likely to be the gene contributing to the majority of the transcript attributed to COR47 (Figure9a). To address whether an alternative spliced product from COR47B (Figure9b) might explain the changes in COR47-like protein expression, we set out first to examine whether any alternatively spliced products were likely. Since only genomic sequences of F. vesca “Hawaii IV” (NCBI) were available, we first sequenced DNA encoding COR47B from all three genotypes used in this study and confirmed that the splice junction sites for the single predicted intron were identical in sequence to that of “Hawaii IV” (data Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 11 of 22 not shown). Using a primer set that flanked the intron, RT-PCR revealed only the fully spliced product, with no evidence for any significant alternatively spliced (or un-spliced) product observed at any time points (Figure9c).

Figure 6. Cont.

Figure 6. Identification of two dehydrins from F. vesca, ‘ALTA’ genotype. (A) SDS-PAGE immunoblot of partially purified dehydrins, probed with combination of anti-XERO2 and anti-COR47 antibodies. Q/FT, Q/W, Q/300 and Q/1000 represent flow-through, a 10 mM NaCl wash, a 300mM NaCl elution and a 1000 mM NaCl elution. (B) Coomassie-stained SDS- PAGE of the Q1000 fraction (see panel A) indicating with boxed regions, the sectors of the gel removed for mass spectroscopic analysis. (C) Amino acid sequence of COR47B with peptides (underlined) obtained by mass spectroscopy (two peptides, coverage = 13%, and the amino acid sequence of XERO2 with peptides (underlined) obtained by mass spectroscopy (five peptides, coverage = 56%).

To further understand the increase of COR47 protein that anticipates the increase in COR47 transcript in ‘ALTA,’ we first considered the unlikely possibility that one of the two isogenes of COR47A and B might be contributing to this interpretation. We considered this unlikely because we only observed a single major antiCOR47 reactive protein band in Western blots (Figure 7B); whereas, if both genes were being expressed, one of them (COR47A) would be predicted to be approximately 50% smaller in mass (see Figure S2). Transcripts derived from COR47A and COR47B were not distinguished in earlier qRT- PCR experiments (Figure 3), so we developed COR47A and B transcript specific primers

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(Table S4) and examined the levels of transcripts in ‘ALTA’ crowns. The COR47A gene was expressed at significantly lower levels (>1000 fold lower) than COR47B. Thus, COR47B seemed likely to be the gene contributing to the majority of the transcript attributed to COR47 (Figure 9a). To address whether an alternative spliced product from COR47B (Figure 9b) might explain the changes in COR47-like protein expression, we set out first to examine whether any alternatively spliced products were likely. Since only Figure 6. Identification of two dehydrins from F. vesca, ‘ALTA’ genotype. (A) SDS-PAGE immunoblot of partially purified genomic sequences of F. vesca “Hawaii IV” (NCBI) were available, we first sequenced dehydrins,Figure 6. Identification probed with of combination two dehydrins of anti-XERO2 from F. vesca and, ‘ALTA’ anti-COR47 genotype. antibodies. (A) SDS Q/FT,-PAGE Q/W, immunoblot Q/300 and of Q/1000partially represent purified dehydrins, probed with combinationDNA encoding of anti-XERO2 COR47B and fromanti-COR47 all three antibodies. genotypes Q/FT, used Q/W, in Q/300this study and Q/1000 and confirmed represent that flow-through, a 10 mM NaCl wash, a 300mM NaCl elution and a 1000 mM NaCl elution. (B) Coomassie-stained SDS-PAGE flow-through, a 10 mM NaClthe wash, splice a 300mMjunction NaCl sites elution for the and single a 1000 predicted mM NaCl intron elution. were (B) identicalCoomassie in-stained sequence SDS to- that of the Q1000 fraction (see panel A) indicating with boxed regions, the sectors of the gel removed for mass spectroscopic PAGE of the Q1000 fraction (seeof “Hawaii panel A) IV” indicating (data notwith shown). boxed regions, Using thea primer sectors setof the that gel flanked removed the for intron, mass spectro- RT-PCR re- analysis. (C) Amino acid sequence of COR47B with peptides (underlined) obtained by mass spectroscopy (two peptides, scopic analysis. (C) Amino acidvealed sequenc onlye the of COR47Bfully spliced with peptidesproduct, (underlined) with no evidence obtained for by anymass significant spectroscopy alternatively (two coverage = 13%, and the amino acid sequence of XERO2 with peptides (underlined) obtained by mass spectroscopy peptides, coverage = 13%, andspliced the amino (or un acid-spliced) sequence product of XERO2 observed with peptides at any (underlined) time points obtained (Figure by9c ).mass spectros- (fivecopy peptides,(five peptides, coverage coverage = 56%). = 56%).

FigureFigure 7. 7.Dehydrin Dehydrin protein protein accumulation accumulation in cold-treatedin cold-treated ‘ALTA’ ‘ALTA’ crowns. crowns. Panel Panel (A), Western (A), Western blot of blot genotype of genotype ‘ALTA’ probed‘ALTA’ withprobed anti-XERO2. with anti-XERO2. Panel (B Panel), Western (B), Western blot of genotype blot of genotype ‘ALTA’ ‘ALTA’ probed withprobed anti-COR47. with anti-COR47. Panel ( CPanel), quantitation (C), quantitation of band of intensityband intensity in Panel in (PanelA) (anti-XERO (A) (anti-XERO 2). Circles 2). Circles represent represent the 35-36 the kDa35-36 band, kDa band, squares squares represent represent the 25 the kDa 25 band. kDa band. Panel Panel (D), Quantitation(D), Quantitation of band of band intensity intensity in Panel in Panel (B) (anti-COR47). (B) (anti-COR47). All signalAll signal quantitation quantitation are expressedare expressed as average as average (and (and standard stand- deviation)ard deviation) values values of three of three biological biological replicates replicates (except (except for the for 1the h time-point1 h time-point which which was was in duplicate) in duplicate) as shown as shown on gelson gels in panelsin panels (A, B(A).,B Replicates). Replicates acclimated acclimated for for 0 h, 0 1h, h, 1 3h, h, 3 8h, h,8 h, 24 24 h andh and 48 48 h, h, 7 d,7 d, 14 14 d d and and 42 42 d d at at 2 2◦ C°C were were separated separated on on 12%12% SDS-PAGESDS-PAGE gel, gel, transferred transferred to to a a nitrocellulose nitrocellulose membrane membrane and and probed probed with with the the respective respective antibody. antibody.

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Figure 8. COR47 (A) and XERO2 (B) transcript and protein levels following LT show distinctive responses in the ‘ALTA’ Figurecrown (crn).8. COR47 Relative (A) and protein XERO2 levels (B) (Figure transcript7) are and directly protein compared levels following to transcript LT show levels distinctive (Figure3). responses in the ‘ALTA’ crown (crn). Relative protein levels (Figure 7) are directly compared to transcript levels (Figure 3).

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FigureFigure 9. 9.COR47B COR47B is is the the major major COR47 COR47 transcript transcript and and is is not not alternatively alternatively spliced. spliced. ((aa)) Comparison Comparison ofof transcripttranscript levelslevels (Cts)(Cts) fromfrom the the distinct distinctCOR47A COR47Aand andCOR47B COR47Bisogenes isogenes during during cold cold treatment. treatment. Speciﬁc Specific primers primers were were developed developed to to distinguish distinguish transcriptstranscripts fromfrom the the two two genes genes (Table (Table S4). S4). ( b(b)) Possible Possible RNA RNA products products from fromCOR47B COR47Bgene. gene. (c(c)) A A single single spliced spliced COR47B COR47B productproduct was was seen seen at at all all cold cold time time points. points.Analysis Analysis ofof qRT-PCRqRT-PCR products products utilizing utilizing primers primers (Figure (Figure S1) S1) to to amplify amplify the the region region surroundingsurrounding the the intron intron of of the theCOR47B COR47Bgene gene indicates indicates the the presence presence of of a a single single major major species species consistent consistent with with a a spliced spliced intron intron (expected(expected 239 239 b.p.) b.p.) compared compared to to an an intron-retained intron-retained product product (expected (expected 397 397 b.p.). b.p.). All All time time points points of of the the cold cold treatment treatment show show aa similarlysimilarly sizedsized splicedspliced product.product. TheseThese representrepresent endpointsendpoints ofof qRT-PCRqRT-PCR reactionsreactions andand thusthus cannotcannot bebe comparedcompared onon aa quantitative level. Left and right panels were results of samples run on different gels. quantitative level. Left and right panels were results of samples run on different gels.

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3. Discussion During development and growth, plants endure abiotic stresses which negatively affect crop yields and survival. The diploid Fragaria genotypes (“wild” or woodland strawberries) show significant variation in cold tolerance, with LT50s varying from −8 to −12 ◦C [24]. Analysis of diploid responses to cold should be useful for understanding more complex genotypes (such as octoploids) for commercial strawberry production. One important goal of the present work was to identify the likely cold responsive transcripts of CBFs in F. vesca. The cold-inducible CBF genes are the primary regulators of cold acclimation and the expression of CBFs is crucial for freezing tolerance in A. thaliana [16,53,62,63]. Increases in CBF transcript levels are detectible within 15 min of transferring plants to low temperature. CBF transcripts are usually at their highest levels by 4 h of LT at 4 ◦C, thereafter gradually decreasing to lower levels, though still signiﬁcantly elevated at 24 h of LT [64,65]. FaCBF1 (octoploid strawberry) and PcCBF1 (sour cherry) were found to be up- regulated within 1 h of exposure to cold, peaked at 4 h, then followed a transient decrease in expression level [66]. The two most likely candidates were identiﬁed by the presence of critical features including and surrounding the DNA-binding (AP2) sites. In addition to FvCBF1E’s very closely related sequence to other characterized CBFs (Figure S1), analysis of the kinetics of cold expression showed a typical CBF cold response. The up-regulation of FvCBF1E was highest in the leaves of the most cold-tolerant genotype ‘ALTA’, compared to the two less cold-tolerant genotypes analyzed in this study. Interestingly, in crowns of the ‘ALTA’ genotype, FvCBF1B.1 transcripts (the most closely related sequence to Arabidopsis CBFs) were detected with levels much higher than the other genotypes (Table S3) but became undetectable following cold treatment (Figure1). The FvCBF1B.1 transcripts were only moderately up-regulated in the crowns of ‘NCGR1363’ and ‘FDP817’ and in the leaves of all genotypes, suggesting that the FvCBF1B.1 gene product contributes much less to cold tolerance in F. vesca tissues. Functional characterization will be necessary to verify the role of FvCBF1E in cold tolerance. To begin to address the question of whether ABA-responsive genes may contribute to cold-stress tolerance in F. vesca, the transcript level of three different F. vesca genes containing the conserved ABRE promoter element were investigated during a 42 d LT treatment. Only the FvGEM transcript, encoding a protein annotated as a GEM-like protein 5-like (NCBI) and orthologous to the GRAM domain family protein in A. thaliana (At5g13200) was found to be regulated in crowns in response to LT treatment (Figure4 ). FvGEM was strongly down-regulated in ‘ALTA’ crowns and was up-regulated in the two lesser cold-tolerant F. vesca genotypes. Transcription of the A. thaliana GEM-homolog (At5g13200), was up- regulated approximately 10-fold in leaves after 24 h of cold treatment (AtGenExpress Visualization Tool [67,68]). The responses of these genotypes suggest that FvGEM may not be suitable as a useful marker for differential LT tolerance in F. vesca. In an earlier study it was suggested that ADH could serve as an important marker for cold tolerance in diploid strawberry [24]. ADH is known to have an important role in other abiotic stress conditions, including hypoxia, and the activity of ADH is vital for plant survival during anaerobic conditions. ADH mRNA accumulated to high levels by 48 h in both crown and leaf tissues from all diploid genotypes (Figure2). The most cold-tolerant diploid strawberry (‘ALTA’) had the greatest fold increase in ADH transcripts levels. The strong and sustained increases in ADH transcript level described here are consistent with the cold-induced protein levels previously shown (ADH protein levels in F. vesca were highly correlated with LT50 (r = −0.86, [24]). These results are also consistent with responses in octoploid strawberry (Fragaria × ananassa) where previous work has shown that during cold-acclimation four ADH protein isoforms accumulated to higher levels in the freezing-tolerant cultivar ‘Jonsok’ compared to the less-tolerant ‘Frida’ and that the protein accumulation peaked after 42 d of LT [5]. As in octoploid strawberry, a key role for elevated levels of ADH contributing to enhanced freezing tolerance is consistent with the ability of ADH to enhance stress survival by alleviating hypoxic conditions caused by ice encasement [69]. Int. J. Mol. Sci. 2021, 22, 6124 15 of 21

A typical plant molecular response to cold stress involves expression of members of a family of genes encoding the dehydrin proteins. High accumulation levels of dehydrin proteins in plants are associated with its freezing tolerance [50,51]. Seven putative F. vesca dehydrins, showing five distinctive structural types (Figure S2), were identified. These dehydrins cluster in two major phylogenetic groups [24] (Figure S3) similar to those in other plant species [37,70]. In this report, we described changes in abundance of both basic and acidic dehydrin transcripts and, after identifying the proteins by mass spectroscopy, one dehydrin representative from each group: XERO2-like, a basic dehydrin and COR47-like, an acidic dehydrin were chosen. FvXERO2 transcripts were highly up-regulated in both “ALTA” crown and leaf tissues from 48 h to 42 d of LT, peaking at 42 d. The most cold- tolerant genotype, ‘ALTA’, accumulated the highest FvXERO2 transcript levels compared to the two less cold-tolerant genotypes ‘NCGR1363’ and ‘FDP817’, similar to results found previously in octoploids [5]. FvXERO2 transcripts, thus, showed strong and consistent increase over the entire exposure to cold. Protein levels also increased, with a slight delay (relative to the XERO2 transcripts), consistent with XERO2 transcripts driving increases in XERO2 protein accumulation. A modest increase in transcript levels of a representative of the acidic dehydrins, FvCOR47, peaked at 48 h followed by a decreased level in accumulation until 42 d. This is similar to the pattern observed in A. thaliana, where COR47 has been shown to be transiently up-regulated during LT and then decreases in expression after 14–21 d of LT [71]. However, it is quite different from the pattern found in octoploid cold-tolerant ‘Jonsok’ where a rapid, but transient cold response accumulation was observed, peaking at 1 d of LT [5]. In the ‘ALTA’ genotype, decreases in transcript levels were incongruous with the increases in COR47 protein levels (Figure8). Possible explanations, such as the alteration of levels of COR47 isoforms or alternative splicing, were explored. The possibility of the COR47A gene contributing significantly at the protein level seemed quite unlikely, as its transcript levels were several orders of magnitude lower than the major isoform. Further, the predicted COR47A protein was smaller and was expected to be readily resolved from the COR47B protein and thus was not included in the COR47B protein quantitation. In terms of alternative splicing, the COR47B gene has only a single predicted intron and no unspliced products and no alternatively spliced products were observed at any LT treatment time point. The mechanism for the post-transcriptional regulation of COR47 must be further explored.

4. Materials and Methods 4.1. Plant Material Plant runners of three F. vesca genotypes (‘ALTA’, ‘FDP817’ and ‘NCGR1363’) were planted in a peat-based potting compost (90% peat, 10% clay) containing 1:5 (v/v) gran- ulated perlite in a 10 cm plastic container. The plants were ﬂow-irrigated twice a week with a solution containing 7.8 mM nitrogen, 1 mM phosphorus and 4.6 mM potassium per liter. The plantlets were propagated in a greenhouse with supplementary light (temperature 18 ± 2 ◦C and 20 h photoperiod) and grown for 42 d. Cold acclimation (CA) was performed by transferring plants to a cold room at 2 ◦C with a short-day photo-period (Supplemental light was provided by high-pressure sodium lamps (SON-T), 10 h light/14 h dark at 90 µmol m−2 s−1). Whole plant leaves and entire crown tissues were harvested and ﬂash frozen in liquid nitrogen after 0 h (untreated), 1 h, 2 h, 3 h, 8 h, 24 h, 48 h, 7 d, 14 d, and 42 d of low temperature treatment. The 0 h untreated control plants were harvested at room temperature while the cold treated plants were harvested in the cold room. Triplicate samples (biological replicates, each replicate containing material from 5 plants) of both leaves and crown tissues from each genotype were harvested for each time point and stored at −80 ◦C until RNA or protein extraction. Int. J. Mol. Sci. 2021, 22, 6124 16 of 21

4.2. Identification of Putative F. vesca ABA-Responsive and Cold-Responsive Genes Potential ABA and cold responsive genes in A. thaliana were identified by BLASTn searches using the ABRE promoter consensus sequence motif (ACGTGGC/T) as a query against the RefSeq A. thaliana database. Selected genes that contained ABREs and that were functionally annotated as cold responsive in Arabidopsis (TAIR; www.arabidopsis.org) were used as query sequences in a second BLASTn search to identify homologous sequences in the F. vesca reference genome (http://www.phytozome.net (accessed on De- cember 2014)). All blast searches were performed using default parameters. Putative F. vesca dehydrin proteins were identified by performing BLASTp searches using the K- segment motif (EKKGIMDKIKEKLPG), which is considered diagnostic for dehydrins, as query against the F. vesca RefSeq Protein Database at NCBI. The blast search parameters were adjusted for small input query sequences. These full-length sequences were then queried against the latest annotation of F. vesca v.4.a2 (ftp://ftp.bioinfo.wsu.edu/species/ Fragaria_vesca/Fvesca-genome.v4.0.a2/ (accessed on 10 September 2020)) for updated gene IDs and BLASTp-based homologies of predicted protein sequences against the plant NR protein database (ftp://ftp.bioinfo.wsu.edu/species/Fragaria_vesca/Fvesca-genome. v4.0.a2/homology/blastp_Fvesca_v4.0.a2_vs_nr.xlsx.gz (accessed on 10 September 2020)). Identified sequences were imported to CLC Genomic Workbench (Qiagen, Hilden, Ger- many) and annotated for the presence of conserved dehydrin sequence motifs, including the Ser-cluster, Y-segment and the K-segment. This was done by creating a local database consisting of the putative F. vesca sequences and searching it with the Y- and K-segment consensus sequences as queries, with a manual supervised curation by one of the authors. A phylogenetic analysis was performed using Clustal Omega to build the multiple sequence alignment, and then a phylogenetic tree was constructed using NJPLOT using the neighbor joining method [72].

4.3. qPCR and Verification of Amplification Products RNA was extracted from 100 mg F. vesca crown and leaf tissues using Spectrum Plant Total RNA Kit (Sigma Aldrich, St. Louis, MO, USA) according to the manufacturer’s instructions. Prior to reverse transcription, traces of residual genomic DNA were removed by digestion of 2 µg RNA with 1 U RNase-free amplification grade DNase I (Invitrogen, Carslbad, CA, USA) in a total reaction volume of 22 µL in accordance with the manufacturer’s instructions. Of 36 representative RNA samples tested, 19 had RIN values of 9–10, 16 had 8–9 and one had a RIN value of 7.8. Gene-specific primers for qPCR were designed for 10 targets (Table S4). Transcript FvPP2A (serine/threonine protein phosphatase 2A), known to be stable to cold treatment in Arabidopsis [73] and verified to be stable in response to cold treatment based on RNASEQ data in both F. ananassa and F. vesca (data not shown), was used for qPCR data normalization. Primers were designed using CLC Genomics Workbench 11 (Qiagen, Hilden, Germany) using the following parameters: primer length 17–26 nt; melting temperature (Tm) 59–62 ◦C; %GC content 30–70; NaCl 50 nM; dNTPs 0.1 mM and MgCl2 1.5 mM. The qPCR amplicon lengths were between 90–180 base pairs. First strand cDNA synthesis was performed with 1 µg DNase I-treated total RNA using 200 U Superscript TM III reverse transcriptase (Invitrogen, Carslbad, CA, USA), primed with 2.5 µM random hexamer primers (pdN6) and 250 ng oligo (dT)18 primers, in a total reaction volume of 20 µL according to manufacturer’s instructions. Transcript levels of each target were analyzed by real time qPCR using EvaGreen® to monitor dsDNA synthesis. Each reaction was composed of 2 µL 5X Hot FIREpol EvaGreen qPCR Mix plus ROX (Solis BioDyne, Tartu, Estonia) and 0.1 µM gene-specific primers in a total reaction volume of 10 µL. qPCR was performed with the AB17500 real time PCR detection system (Applied Biosystems, Foster City, CA, USA) using the following thermal cycling parameters: 50 ◦C for 2 min, 95 ◦C for 12 min, then 40 cycles of 95 ◦C for 30 s, 60 ◦C for 1 min, 72 ◦C for 1 min, and a final dissociation step. ROX was used as the internal passive reference dye to normalize the fluorescent reporter signal. Post-qPCR dissociations were performed to verify a single peak and at least one amplicon from each primer set was sequenced to verify Int. J. Mol. Sci. 2021, 22, 6124 17 of 21

the appropriate target. PCR efficiencies (80–100%) were determined by a linear regression method (utilizing the LinRegPCR program; [74]), and the average PCR efficiency values for each respective amplicon group were utilized. The expression level of each transcript was normalized to the reference gene FvPP2A used as internal loading control. Relative transcript levels compared to the control sample (0 h untreated plant) were calculated using the Pfaffl method [75].

4.4. Western Blotting Total protein was extracted from the same experimental plant material as that used for the transcript analyses to investigate dehydrin protein accumulation. Powders from crown and leaf tissue (50 mg) were homogenized in hot 2x SDS-PAGE sample buffer (SSB) containing 100 mM DL-dithiothreitol (DTT), 1.5 M Tris-HCl (pH 8.5), 2% glycerol, 2% SDS (w/v), 2% mercaptoethanol and 1x protease inhibitor cocktail (Roche Complete TM protease inhibitor cocktail; Roche, USA). Protein concentrations were estimated using the Amino Black method [76]. Three biological replicates (each of which were composed of 5 individual plants) obtained from each time-point and from all three genotypes were analyzed by Western blotting with a primary antibody raised against the K-sequence motif, which is diagnostic of dehydrin proteins in plants [36–38], anti-AtXERO2 or anti-AtCOR47 primary antibodies as described in [35]. Secondary antibody was anti-rabbit IgG (1:8000, Alexa Fluor 790). After appropriate washing, an image was taken using a Li-Cor Odyssey CLx Imager (169 um resolution) and quantitated using Image Studio Lite Ver 5.2. The relationship between ﬂuorescence vs protein levels were linear up to approximately 10 µg total protein extract (Figure S5). Protein loads for all Western blot analysis were kept at or below 10 µg.

4.5. Dehydrin Extraction, Fractionation and Identification by Mass Spectrometry Tissue from 42 d cold treated ‘ALTA’ crown tissues (1.5 g fresh weight) was extracted in a denaturing buffer, as described for Western blots. This rigorous extraction method was necessary for quantitative removal of dehydrins from crown tissues. The homogenates were then centrifuged for 10 min at 10,000× g to remove debris and 5 volumes of 0.1 M Ammonium Acetate in 100% Methanol were added to the supernatants. The supernatants were stored overnight at −20 ◦C, then centrifuged for 10 min at 10,000× g. The pellets were resuspended in 0.2% (w/w) Triton X-100 and heat-treated in an 80 ◦C water bath for 10 min. The heat-treated samples were then centrifuged for 10 min at 10,000× g and supernatants were stored at −80 ◦C. The Triton X-100 supernatant was diluted 10-fold with 10 mM Tris-HCl buffer, pH 8.5 (at 4 ◦C), and loaded at 0.5 mL.min−1 onto a 50 mL packed bed volume of DEAE-Sepharose (Amersham-Pharmacia Biotech, Uppsala) anion-exchange column. The proteins were eluted with a linear 10 to 300 mM NaCl gradient, followed by a 1 M NaCl elution generated by a Waters 650E Advanced Protein Purification System (Milli- pore, Bedford, MA, USA). Fractionated protein samples were separated by 12% SDS-PAGE gels as the final purification step. Western blots were performed and Coomassie-stained dehydrin protein bands corresponding to apparent mass of 35–37 kDa (later identified as XERO2; XP_004287863.1), 65–70 kDa (later identified as COR47; XP_011459083.1), or 25 kDa (unsuccessfully identified; note region was excised even though no visible Coomassie- stainable band was present) from duplicate gels were excised with a sterile blade. The gel bands were destained, reduced with 10 mM DTT in 10 mM ammonium bicarbonate and then alkylated with 55 mM iodoacetamide (prepared in 10 mM ammonium bicarbonate). Alkylated samples were digested by trypsin (Promega) overnight at 37 ◦C. Digested peptides were sequentially extracted from the gel spots with: (1) 50% ACN/49.9% H2O/0.1% TFA; and then (2) 99.9% ACN/0.1% TFA. The combined extracts of tryptic peptides were injected into a C18 column (TSKgel ODS-100 V, 3 µm, 1.0 mm × 50 mm). Peptides were eluted with a linear gradient from 5 to 35% acetonitrile (in water with 0.1% FA) developed over 60 min at room temperature, at a flow rate of 50 uL.min−1, and the effluent was electro-sprayed into an LTQ mass spectrometer (located at the Indiana University Purdue Int. J. Mol. Sci. 2021, 22, 6124 18 of 21

University, Indianapolis Proteomic Core Facility). The acquired data were searched against NCBI protein sequence database of F. vesca (http://www.ncbi.nlm.nih.gov/, 57918 entries (accessed on 11 July 2016)) using SequestTM algorithms.

5. Conclusions The cold responsive CBF has been tentatively identiﬁed as FvCBF1E (XP_004298771.1). Interestingly, the similarity of changes of cold-induced CBF levels in the highly cold- tolerant and less tolerant genotypes suggest that the differential cold sensitivity amongst the genotypes may be due to CBF-independent events. The examination of the various transcripts reveals a complexity of both temporal and genotype responses to cold. The changes in transcript levels shown here, together with protein changes [24] are consistent with an important role of ADH in cold tolerance. Dehydrins likely also play an important role in cold acclimation and protein levels correlate well with cold tolerance among the genotypes. In the case of COR47B, it appears that post-translational regulation is important for control of dehydrin protein levels. While requiring further investigation, these ﬁndings suggest that post-transcriptional cold-regulation of protein levels may be an important contributor to cold tolerance mechanisms.

Supplementary Materials: The following are available online at https://www.mdpi.com/article/10 .3390/ijms22116124/s1, Figure S1: Phylogenetic analysis showing the relationship between F. vesca (Fv) putative CBFs (AP2 containing) and A. thaliana (At), S. lycopersicum (Sl), and G. max (Gm) CBF/DREBs. Figure S2: Annotation of F. vesca dehydrin proteins and their Y-, K-, and S-segments. Figure S3: Phylogenetic analysis showing the relationship between ten A. thaliana dehydrins and the seven putative F. vesca dehydrin proteins identified in this study. Figure S4: Corresponding changes of FvXERO2 and FvCOR47 transcript and protein in leaves and crowns of three F. vesca genotypes in response to LT. Figure S5: Quantitation of FvCOR47 is linear over a 10-fold range of protein (maximum of 10 µg). Table S1: (A) Alignment of the CBF candidates; their AP2 domain and immediately flanking regions are compared to the CBF consensus (JAGLO). (B) Amino acid sequences used for phylogenetic analysis of CBFs. (C) Amino acid sequences used for phylogenetic analysis of Dehydrins. Table S2: Fragaria vesca and other genes relevant to this study. Table S3: Summary of threshold Cts for all targets under non-acclimating conditions (0 h in the cold). Table S4: qRT-PCR primers used in this study. Author Contributions: J.D., R.C.W., M.A. and S.R. conceived and designed research plan; plants were grown and harvested in the laboratory of J.D., S.R. performed SDS-PAGE and W. blots; Z.D., N.O. and R.N. performed RT-PCR and qRT-PCR; V.M. and N.O. performed initial informatic analysis of FvCBFs; I.F. performed dehydrin purification, prepared and interpreted mass spectrometric data; J.D., R.C.W., S.R., Z.D., I.F., N.O. and R.N. analyzed the data; I.F. and S.R. co-wrote the manuscript with M.A. and R.C.W. performing key editing functions. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by The Research Council of Norway (NFR # 244658) and Graminor Breeding Ltd. (Project #244658/E50). Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: Data is contained within the article or Supplementary Material. Acknowledgments: This work was supported by The Research Council of Norway (NFR # 244658) and Graminor Breeding Ltd. (Project #244658/E50) and a research stipend from INN University to R.N., and R.C.W. Conflicts of Interest: The authors declare no competing interests.

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